Method of altering sphingolipid metabolism and detecting fumonisin ingestion and contamination

ABSTRACT

This invention provides a method of altering the metabolism of sphingolipids in a cell comprising contacting the cell with a fumonisin, or an analog thereof. The invention also provides a method of detecting the consumption of a fumonisin or a fumonisin analog in a subject comprising (A) detecting, in a sample from the subject, the state of the metabolic pathway of sphingolipids and (B) comparing the state of the metabolic pathway to that of a normal subject, the presence of a change in the state of the metabolic pathway indicating the consumption of a fumonisin or a fumonisin analog. Also provided is a method of detecting the presence of a fumonisin or fumonisin analog contamination in a sample from a food or feed comprising detecting a reaction of the metabolic pathway of sphingolipids, the presence of the reaction indicating the presence of a fumonisin or fumonisin analog contamination. Furthermore, novel fumonisin analogs and compositions comprising fumonisins and fumonisin analogs are provided.

The following invention was made through support from grant number GM33369 from the National Institutes of Health, grant number DCB 8710283from the National Science Foundation and grant number CRIS6612-42000-004 to the United States Department of Agriculture. TheUnited States government has rights in the invention.

Throughout this application, various publications are referenced. Thefull citation of these publications is provided in a bibliographyimmediately before the claims. The disclosures of these publications arehereby incorporated by reference to more fully describe the state of theart at the time the invention was made.

BACKGROUND OF THE INVENTION

Fusarium moniliforme (Sheldon) is one of the most prevalent fungi onmaize, other grains, and agricultural commodities in the United Statesand throughout the world (1). Culture materials from certain isolates of(and grains naturally contaminated with) F. moniliforme have been shownto be toxic and carcinogenic for animals (2-5); furthermore, consumptionof contaminated maize has been correlated with esophageal cancer inareas of southern Africa, China, and other countries (6-8). Severalmycotoxins, termed fumonisins, have been isolated from extracts of F.moniliforme (9) and naturally contaminated corn (10,11). Fumonisin B₁has been shown to cause equine leucoencephalomalacia (12), porcinepulmonary edema (13), and promotion of liver tumors in rats (14). Recentsurveys indicate that high levels of fumonisin B₁ are present on UnitedStates feeds associated with field cases of these animal diseases (15).

The molecular mechanism of action of the fumonisins is not known.However, this invention provides the discovery that these compounds beara remarkable structural similarity to sphingosine, the long-chain(sphingoid) base backbone of sphingomyelin, cerebrosides, sulfatides,gangliosides and other sphingolipids. Sphingolipids are thought to beinvolved with the regulation of cell growth, differentiation, andneoplastic transformation through participation in cell-cellcommunication and cell-substratum interactions, and possibleinteractions with cell receptors and signalling systems (16-20). Thisinvention provides that fumonisins and fumonisin analogs target thedisruption of sphingosine metabolism and that fumonisins and fumonisinanalogs inhibit de novo sphingolipid biosynthesis in animals. Thisinvention further identifies the reactions catalyzed by ceramidesynthase as important sites for altering sphingolipid metabolism. Thiscore discovery of the biochemical action of fumonisins provides a meansto treat the many diseases associated with sphingolipid metabolism aswell as a means for early detection of fumonisin contamination oringestion.

SUMMARY OF THE INVENTION

This invention provides a method of altering the metabolism ofsphingolipids in a cell, comprising contacting the cell with a fumonisinor an analog thereof. Also provided is a method of treating an abnormalcondition in a subject associated with an alteration in sphingolipidmetabolism comprising administering a fumonisin, or an analog thereof,to the subject in a metabolism altering amount. In addition, acomposition comprising a fumonisin, or an analog thereof, in apharmaceutically acceptable carrier is provided. As used herein "afumonisin" only includes those fumonisins with the metabolism alteringactivity.

Also provided is a method of treating an abnormal condition in a subjectassociated with the ingestion of a fumonisin, or an analog thereof,which results in the depletion of a sphingolipid in the subject,comprising adding an amount of the sphingolipid to the subjectsufficient to treat the condition. Further provided is a method oftreating an abnormal condition in a subject associated with theingestion of a fumonisin, or an analog thereof, which results in theaccumulation of a sphingolipid, comprising adding an amount of aninhibitor of the sphingolipid to the subject sufficient to treat thecondition.

The invention also provides a method of detecting the consumption of afumonisin, or an analog thereof, in a subject comprising (A) detecting,in a sample from the subject, the state of the metabolic pathway ofsphingolipids and (B) comparing the state of the metabolic pathway tothat of a normal subject, the presence of a change in the state of themetabolic pathway indicating the consumption of a fumonisin or afumonisin analog. Also provided is a method of detecting the presence ofa fumonisin or fumonisin analog contamination in a sample from a food orfeed comprising detecting a reaction in the metabolic pathway ofsphingolipids, the presence of the reaction indicating the presence of afumonisin or fumonisin analog contamination.

Furthermore, the invention provides a method of diagnosing an abnormalcondition in a subject associated with an alteration in sphingolipidmetabolism comprising detecting the state of the metabolic pathway ofsphingolipids in a subject and comparing the state of the metabolicpathway to that of a normal subject, the presence of a change in thestate of the metabolic pathway indicating the presence of an abnormalcondition.

Finally, this invention provides for novel fumonisin analogs andcompositions comprising fumonisins and fumonisin analogs.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. shows structures of sphingosine, Alternaria toxins, fumonisinanalogs and fumonisins B₁ and B₂.

FIG. 2 shows sphingosine biosynthesis and inhibition by fumonisin B₁.Free long-chain bases (3-ketosphinganine, sphinganine, and sphingosine)have not been found to accumulate as detectable intermediates of thispathway under normal conditions; therefore, sphingosine formation isassessed by hydrolyzing the N-acyl products and analyzing the labelledlong-chain bases as depicted in this figure. The chromatogramsillustrate the labelling of sphinganine and sphingosine obtained by acidhydrolysis of the lipid extracts from hepatocytes incubated for 2 h with[¹⁴ C]serine with and without 1 μM fumonisin B₁. The large amount ofradiolabel that migrates with the solvent front is primarily fattyacids.

FIG. 3 shows time and concentration dependence of the inhibition of [¹⁴C]serine incorporation into [¹⁴ C]sphingosine by fumonisins B₁ and B₂.Panel A: relative amounts of [¹⁴ C]sphingosine formed from [¹⁴ C]serineby hepatocytes exposed to no fumonisin B₁ (none), to 1 μM fumonisin B₁and [¹⁴ C]serine for 2 h (2 h), upon pretreatment with 1 μM fumonisin B₁for 16 h followed by incubation with [¹⁴ C]serine for 2 h, and to 1 μMfumonisin B₁ or B₂ and [¹⁴ C]serine for 16 h. Panel B: concentrationdependence of fumonisin B₁ inhibition of [¹⁴ C]serine incorporation into[¹⁴ C]sphingosine by rat liver hepatocytes. The assay conditions forthis experiment were the same as for the 2 h incubations described inFIG. 2, and each data point represents the mean ±SD for triplicatedishes of hepatocytes. All of the groups were significantly differentfrom the untreated control (P<0.05).

FIG. 4 shows increases in sphinganine upon incubating hepatocytes withfumonisin B₁. A: Amounts of label detected in [¹⁴ C]sphinganine; theconditions for these analyses were the same as in FIGS. 2 and 3; B: Massamounts of sphingosine and sphinganine after incubation of hepatocyteswith fumonisin B₁ for 2 h, with analysis of the free long-chain bases byHPLC (28); C: HPLC profile of the free long-chain bases of rat liverhepatocytes after incubation with 1 μM fumonisin B₁ for 2 h (forcomparison with the usual HPLC profile of untreated liver see reference28); D: Inhibition of [³ H]sphingosine conversion to ceramides byfumonisin B₁. Hepatocytes were incubated with 1 μM fumonisin B₁ for 1 h,then approximately 1 μCi of [³ H]sphingosine was added and the cellswere incubated for the times shown. The lipids were extracted andanalyzed by TLC as described in the text. Asterisks designate the groupsthat are different from control (no fumonisin treatment) with P<0.05.

FIG. 5 shows inhibition of ceramide synthase by fumonisin B₁ in vitro.

FIG. 6 shows inhibition of de novo sphingosine production uponincubation of rat hepatocytes with Alternaria toxins (AAL toxins).

FIG. 7 shows the concentration of sphingosine, sphinganine and theirratio over time in the plasma of a pony fed fumonisin-contaminated food.

FIG. 8 shows the plots of AST/SGOT and sphinganine to sphingosine ratioover time in the plasma of a pony fed fumonisin-contaminated feed at 15and 22 ppm fumonisin B₁.

FIG. 9 shows the synthesis of a fumonisin analog.

FIG. 10 shows reaction schemes which can be employed to control thestereochemistry of the 2-aminopenta-3,5-diol head group during thesynthesis of a fumonisin analog.

FIG. 11 shows various reaction schemes that can be employed to controlthe stereochemistry of the aminotriol head groups present in Alternariatoxins or fumonisin analogs similar to Alternaria toxins.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides the core discovery that fumonisins, and theiranalogs, affect the biosynthetic pathway of sphingolipids. Fumonisinsand their analogs are generally referred to collectively as "fumonisin"or "fumonisins" herein. However, analogs may also be specificallyaddressed at various times in the specification. "Purified fumonisin"means a fumonisin separated from at least some contaminants associatedwith fumonisins occurring in nature.

Thus, the present invention provides a method of altering the metabolismof sphingolipids in a cell comprising contacting the cell with ametabolism altering amount of a fumonisin, or an analog thereof. Indetermining the precise mechanism through which fumonisins act, it hasbeen discovered that the alteration in metabolism is effected by thefumonisin, or an analog thereof, binding to ceramide synthase in theconversion of sphinganine to dihydroceramide. Additionally, theinvention provides that the alteration in metabolism can be effected byfumonisin, or an analog thereof, binding to ceramide synthase in theconversion of sphingosine back to ceramide. However, the fumonisinscould act on various other aspects of the sphingolipid biosyntheticpathway and thus are not limited by the particular pathway so long assphingolipid metabolism is altered.

As used herein, "ceramide synthase" means an enzyme or enzymes which addin N-acyl linkage to sphingosine, sphinganine and other long-chainbases. Thus, both sphinganine N-acyltransferase, which catalyzes theconversion of sphinganine to dihydroceramide, and sphingosineN-acyltransferase, which catalyzes the conversion of sphingosine toceramide, are referred to as ceramide synthase herein. As also usedherein, "analog of dihydroceramide or ceramide" means a compound whichcan be catalyzed by a fumonisin or an analog.

This invention demonstrates that fumonisins can alter the metabolism ofsphingolipids. The invention provides the discovery that fumonisins havea structure similar to sphingosine. Thus, the activity of the fumonisinslikely relates to this structural similarity. In this regard, thefumonisins and sphingosine share a 2-amino-3-ol head group and possessbackbone carbon chains of approximately equal size. However, sphingosineis hydroxyl-substituted at carbon 1 while fumonisin B₁ and B₂ arehydroxyl-substituted at carbon 5. In addition, the fumonisins possesspolycarboxylate moieties attached to the tail of the backbone carbonchain. Thus, these similarities and differences between sphingosine intheir head groups and tails are responsible for the activity of thefumonisins. Moreover, one merely needs to make various analogs based onthese principles and test the analogs to determine if they havesphingolipid metabolism affecting activity. Such making can be doneusing techniques set forth herein. Modifications on these techniques toobtain additional analogs would be standard. Likewise, these compounds,once synthesized, could be tested for activity using the methods setforth below

Thus, "fumonisin analog" as used herein is given a function as well asstructural definition. "Fumonisin analog" means any compound which hasstructural similarity to fumonisin B₁, B₂, B₃ or B₄ and can alter themetabolism of sphingolipids. By structural similarity to a fumonisin, wemean analogs comprised of three basic units as given in FIG. 1: (a) a1-aminobutan-2-ol head group which may be further substituted bysubstituents X, Y and IV; (b) a Spacer Group consisting of an alkyl,hydroxyalkyl or dihydroxyalkyl chain (straight or branched); (c) ahydrophilic tail (Z-W) in which Z contains an oxygen or nitrogen atomwhich can be further functionalized to attach mono- or polycarboxylatemoieties (W). In this structure, the Spacer Group=alkyl, hydroxyalkyl ordihydroxyalkyl (straight chain or branched, C₁ -C₂₀); Z=H, O, NH, NQ,NQC(O), NHC(O), CO₂, C(O)NH, C(O)NQ, wherein Q is an alkyl (straightchain or branched, C₁ -C₆); W=no substituent, H, alkyl (straight chainor branched, C₁ -C₆), aryl (phenyl, substituted phenyl such assubstitution with alkyl (straight chain or branched, C₁ -C₆) or halo),C(O)(CH₂)_(n) CO₂ H (where n=1-6), C(O) (CH₂)_(n) CW'₂ CO₂ H, where W'is selected independently from H, alkyl (straight chain or branched, C₁-C₆), aryl (phenyl, substituted phenyl such as substitution with alkyl(straight chain or branched, C₁ -C₆) or halo), (CH₂)_(n) CO₂ H andn=1-6; X=H, methyl, CH₂ OH (and esters thereof), CH₂ NQ'₂ (where Q' isselected independently from H, alkyl (straight chain or branched, C₁-C₂₀), acyl (C(O)Q" where Q" is an alkyl, straight chain or branched, C₁-C₂₀)); and V and Y=H or OH (and esters thereof). Also, explicitlyincluded in the term "fumonisin analog" are the Alternaria toxins (AAL,toxins) as given in FIG. 1 and discussed in reference 33 and, byinference, similar compounds produced by other plants and fungi.Specific structures of various analogs and general synthesis methods areset forth below and in FIGS. 1, 9 and 10.

"Altering the metabolism" as used herein means either an increase or adecrease in the concentration of compounds formed in the sphingolipidbiosynthetic pathway depending on what point in the pathway is affected.Typically, since ceramide synthase (sphinganine-N-acyltransferase) isgenerally bound by the fumonisin or fumonisin analog, and ceramidesynthase (sphinganine-N-acyltransferase), which can also be bound byfumonisins, catalyzes the conversion of sphinganine to dihydroceramidein the pathway, the concentration of any compound above the site ofaction can be increased and the concentration of any compound below thesite of action can be decreased using the fumonisin or fumonisin analog.However, the amounts of various compounds of the pathway can depend onthe time period after inhibition or their relationship to other relatedpathways. For example, a fumonisin can bindsphingosine-N-acyltransferase, which would increase the amount ofsphingosine by preventing the conversion back to ceramide and afumonisin can also bind ceramide synthase(sphinganine-N-acyltransferase), which would decrease the amount ofsphingosine by decreasing the amount of ceramide which can be convertedto sphingosine. Thus, the fumonisin can cause both the increase and/ordecrease of sphingosine, depending on which pathway is dominant. Suchincreases or decreases at various times after fumonisin ingestion can beroutinely determined by methods utilized herein.

"Contacting the cell" as used herein means the fumonisin is administeredin such a way that the fumonisin can enter the cell and affect thesphingolipid biosynthetic pathway within the cell. As shown below, thecell can be in a human or a nonhuman animal. Such in vivo activity isnot only shown, but would be expected, because the nature of thesphingolipid metabolism would not differ fundamentally in vivo.

"Metabolism altering amount" as used herein means a quantity of afumonisin capable of effecting a change in the sphingolipid metabolismin cells. Naturally, this amount can vary depending on the environmentin which the cells are contained. For in vivo administration, theprecise quantity of the fumonisin to elicit the desired effect can bedetermined by standard administration and monitoring until optimaldosage is determined. Such amounts can vary from subject to subjectdepending on size and condition of the subject.

As discussed above, the invention also provides a method of treating anabnormal condition in a subject associated with an alteration insphingolipid metabolism. This method comprises administering afumonisin, or an analog thereof, to the subject in a metabolism alteringamount. The subject can be a human or a nonhuman animal and the abnormalcondition can be caused by numerous disorders in sphingolipidmetabolism. For example, the condition can be an excess of sphingosineresulting in Neimann-Picks syndrome, Tay-Sachs disease, a neoplasticcondition or toxicity. In a given condition, the effects of fumonisinmay be complex. For example, in a neoplastic condition, the fumonisincan, due to its structural similarity to sphingosine, prevent theformation of a sphingolipid required for growth of the cells and canalso act as an inducer of differentiation and thus treat the neoplasticcondition.

The invention also provides a method of treating an abnormal conditionin a subject associated with the ingestion of a fumonisin or fumonisinanalog which results in the depletion of a sphingolipid in the subject.This method comprises adding an amount of the sphingolipid to thesubject sufficient to treat the condition. As can be seen by the pathwayset forth in the figures, in general, compounds that occur in thebiochemical pathway of sphingolipids after sphinganine can be depletedif sphinganine is not converted to dihydroceramide due to a fumonisin.Thus, adding the depleted compound or alternate compounds bypasses theblocked step. Similarly, fumonisins can bind ceramide synthase toprevent the conversion of sphingosine to ceramide and lead to anincrease in sphingosine or a decrease in ceramide. Thus, adding thedepleted compound or alternate compounds can also bypass this blockedstep. Such conditions which can be effectively treated by this methodcan be determined by administering the compound to subjects suspected ofsuffering such conditions. Likewise, an administration regimen can beoptimized by routine testing.

Also provided is a method of treating an abnormal condition in a subjectassociated with the ingestion of a fumonisin or a fumonisin analog whichresults in the accumulation of a sphingolipid comprising adding anamount of an inhibitor of the sphingolipid to the subject sufficient totreat the condition. This method can be effective since compounds priorto dihydroceramide, especially sphinganine, can accumulate ifsphinganine is not converted to dihydroceramide. Likewise, compoundssuch as sphingosine can accumulate if sphingosine is not converted toceramide. Regimens can be determined as discussed above.

In treating an abnormal condition, the amount of fumonisin, or an analogthereof, which is administered can vary. Preferably the amount isbetween about 5 and 500 mg, especially between about 25 and 75 mg whenfumonisin B₁ or B₂ is utilized or an analog with a similar activity. Thecompounds according to the present invention are included in thepharmaceutically acceptable carrier in an amount sufficient to exert atherapeutically useful effect without exhibiting adverse toxic effectson the patient treated. Pharmaceutically compatible binding agentsand/or adjuvant materials may also be included as part of thecomposition. The active materials can also be mixed with other activematerials that do not impair the desired action and/or supplement thedesired action. In general, the ratio of the fumonisin or the analog tothe carrier is determined by the rate of administration and can beoptimized using routine procedures.

It will be appreciated by those skilled in the art that the effectiveamount of a compound or formulation containing the compound required totreat an individual will vary depending on a number of factors,including whether the fumonisin or the analog is administered, the routeof administration, the nature of the condition being treated and the ageand condition of the patient. The desired dose may be given in a singledose or as divided doses administered at appropriate intervals, such astwo, three, four or more sub-doses per day. Thus, these compounds ortheir pharmaceutically acceptable derivatives can be convenientlyadministered by a convenient route of administration, such asparenteral, including intramuscular, subcutaneous and intravenous; oral;rectal; nasal; vaginal or by inhalation.

The invention also provides a composition comprising a fumonisin, or ananalog thereof, in a pharmaceutically acceptable carrier. The carriercan be any carrier suitable for administration to a subject, forexample, buffered saline. The fumonisin, or an analog thereof, can bepresent in an amount between 5 and 500 mg. Especially effective is anamount between 25 and 75 mg.

The invention also provides a method of detecting the consumption of afumonisin or a fumonisin analog in a subject comprising (A) detecting,in a sample from the subject, the state of the metabolic pathway ofsphingolipids and (B) comparing the state of the biosynthetic pathway tothat of a normal subject, the presence of a change in the state of thebiosynthetic pathway indicating the consumption of a fumonisin. Thechange in the metabolic pathway can, for example, be an increase insphinganine or a decrease in a compound following sphinganine in thepathway or an increase in sphingosine. The discovery of the fact thatfumonisins act on the sphingolipid biosynthetic pathway allows for easydiagnostics to be established based on pathway changes in a subject suchas a horse or a human. This method provides a means to constantlymonitor humans or valuable farm animals to detect fumonisin or fumonisinanalog intake, such as AAL toxins, before the fumonisin or analog causesa lethal effect.

A method of detecting the presence of a fumonisin or fumonisin analogcontamination in a sample from a food or feed is also provided. Themethod comprises detecting a reaction of the metabolic pathway ofsphingolipids, the presence of the reaction indicating the presence of afumonisin contamination. For example, the reaction can be the preventionof the conversion of sphinganine, or an analog thereof, todihydroceramide or an analog thereof, or the conversion of sphingosine,or an analog thereof, to ceramide, or an analog thereof, by ceramidesynthase. However, again based on the core knowledge of the effect of afumonisin on sphingolipid biosynthesis, related means of detecting thefumonisin can be established. The invention also provides a method ofdetecting the presence of a fumonisin contamination in a sample from afood comprising detecting a reaction of the metabolic pathway ofsphingolipids, the presence of the reaction indicating the presence offumonisin contamination. For example, the reaction is the conversion ofsphingosine to ceramide or an analog thereof by ceramide synthase.

Finally, the invention provides a method of diagnosing an abnormalcondition in a subject associated with an alteration in sphingolipidmetabolism comprising detecting the state of the metabolic pathway ofsphingolipids in a subject and comparing the state of the metabolicpathway to that of a normal subject, the presence of a change in thestate of the metabolic pathway indicating the presence of an abnormalcondition. For example, an increase in sphinganine can indicateincreased risk of esophageal cancer. Such a change can be detected bythe following methods listed below, and abnormal conditions other thanesophageal cancer can be determined by using such methods. Theconditions can result from an ingestion of a fumonisin or a fumonisinanalog such as AAL toxins and include, for example, neoplasticconditions, toxicity, Neimann-Picks syndrome and Tay-Sachs disease.

EXPERIMENTAL PROCEDURES

All compounds listed in this application are commonly used and availableand any abbreviation utilized is readily known to the skilled artisan.

Inhibition of Ceramide Synthase Activity in Rat Liver Microsomes andReduced Conversion of [³ H] Sphingosine to [³ H] Ceramide by IntactHepatocytes

Materials--Fumonisin B₁ and B₂ were purchased from Division of FoodSciences and Technology, CSIR, Pretoria, South Africa. Tissue culturemedia were purchased from Gibco (Grand Island, N.Y.) and collagenase(Type IV), collagen (Type III), and palmitoyl-CoA were from Sigma (St.Louis, Mo.). Matrigel (from Engelbreth-Holm-Swarm (EHS) mouse tumor)were obtained from Collaborative Research Inc. (Bedford, Mass.) The[2-¹⁴ C] serine and the [³ H]sphingosine (prepared by catalyticexchange) were from New England Nuclear (Boston, Mass.). Thesphingolipid standards were purchased from Sigma, or preparedsynthetically (21).

Hepatocyte culture--Hepatocytes were prepared by a collagenase perfusionmethod, as known in the art as described in (22), from maleSprague-Dawley rats (125 to 200 g) fed a chow diet (Ralston Purina, St.Louis, Mo.) ad libitum. The hepatocytes were isolated using carefullyaseptic procedures and plated in 60 mm tissue culture dishes coated withcollagen for short term (<24 h) or with Matrigel for longer term (3 to 4d) experiments. After plating for 4 h, the medium was changed to 2 ml ofDMEM (with 3.7 g/L NaHCO₃) containing 10 μg/ml of insulin, 6.1 g/L ofpenicillin, 10 g/L of streptomycin sulfate, and varying concentrationsof fumonisins (added from a 1 mM solution in Delbecco's phosphatebuffered saline). The cells were maintained in a tissue cultureincubator at 37° C. and an atmosphere of 5% CO₂ ; the medium was changedevery 24 h. Cell viabilities were assessed by the ability of the cellsto exclude 0.1% trypan blue, and were greater than 90%.

Lipid analyses--The de novo biosynthesis of sphingosine was measured, asknown in the art as described in (23,24). Briefly, cells that had beenexposed to fumonisins for 0 to 4 d were changed to new medium containing10 μCi of [¹⁴ C]serine (25 mCi/mmol) in 2 ml of serine-free DMEM (Gibco)in the presence or absence of fumonisin. After incubation for 2 or 16 h,they were scraped from the dishes, 25 μg of carrier sphingosine andsphinganine were added, and the lipids were extracted and acidhydrolyzed to liberate the free long-chain bases using conditions thatgive optimal hydrolysis with minimal decomposition of the labilelong-chain bases, as known in the art as described in (23-25). Thesphinganine and sphingosine were separated by thin-layer chromatographyon silica gel H (Brinkman) plates developed with CHCl₃ :methanol:2N NH₄OH (40:10:1, v/v/v) and the radiolabelling profile (shown in FIG. 2) wasdetermined by radiometric scanning of the plates with a Bioscan System200 Imaging Scanner. Sphingosine and sphinganine were visualized usingninhydrin, which also revealed any acid decomposition products, whichmigrate as a ninhydrin-positive material near the solvent front (whenseen, the data were not considered reliable). The appropriate regions ofthe chromatoplate were scraped, placed in scintillation vials, and 0.2ml of water and 4 ml of a detergent based scintillation cocktail wereadded. After several hours, the samples were counted with theappropriate corrections for quenching. In our experience (23,24,26,27),these procedures give a good representation of the total amount of labelin the long-chain base moieties of cellular sphingolipids.

To measure the relative activity of ceramide synthase in intacthepatocytes, the cells were incubated with 1 μM fumonisin B₁ for 1 h,then 1 μCi of [³ H]sphingosine was added and the cells were incubatedfor varying times. The [² H]sphingosine was freshly purified because itdecomposes rapidly to a material with an R_(f) similar to that ofceramides on TLC. The [³ H]sphingosine was diluted to 1 Ci/mmol andapplied to a small silica gel column (Unisil, Clarkston, Ill.) inchloroform. The column was washed with 10-column volumes of chloroform,the [³ H]sphingosine was eluted with methanol and the purity confirmedby TLC. The [³ H]sphingosine was transferred to a small test tube, thesolvent was evaporated under N₂, a small volume of culture medium wasadded immediately, and the tube was sonicated for a few minutes using abath-type sonicator. Aliquots of this solution were added to the cells,and at the end of the specified incubation period the cells were scrapedfrom the dish, 25 μg of unlabelled ceramide carrier was added, and thelipids were extracted as described above. The [³ H]ceramides wereseparated from [³ H]sphingosine using silica gel H TLC plates developedwith diethyl ether:methanol (99:1, v/v), visualized with I₂ vapor, andthe amount of radiolabel was determined by scintillation counting

Mass measurements of the long-chain bases were conducted by HPLC, as isknown in the art as previously described in (28), with C20-sphinganineas an internal standard. To quantitate total sphingolipids, the extractswere acid hydrolyzed before preparing the samples for HPLC andcorrected, if necessary, for any losses during hydrolysis or extractionby spiking replicate samples with a known amount of ceramide orsphingomyelin.

In vitro assays of serine palmitoyltransferase and ceramide synthase. Amicrosomal fraction was isolated from rat liver and assayed for serinepalmitoyltransferase, as is known in the art as described in (24,29),and ceramide synthase, as is known in the art as described in (30), inthe presence of varying concentrations of fumonisin B₁. The assaymixture for ceramide synthase contained 1 μM [³ H]sphingosine, 25 mMpotassium phosphate buffer (pH 7.4), 0.5 mM dithiothreitol, 200 μMpalmitoyl-CoA, and approximately 0.2 mg of microsomal protein in a totalvolume of 0.1 ml, which were found to be optimal assay conditions. Thereaction was initiated by adding palmitoyl-CoA and, after incubation at37° C. for 15 min, the products were extracted, resolved by TLC, andquantitated as described above. Background counts were subtracted usingdata from identical assays that omitted palmitoyl-CoA.

Other methods--The incorporation of radiolabel into other lipids wasdetermined by separating the phospholipids by TLC using silica gel Hplates developed with CHCl₃ :methanol:formic acid:water (56:30:2:1,v/v/v/v), visualization of the spots and standards with I₂ vapor, andquantitation by scintillation counting. The amount of label in fattyacids was estimated using samples that had been incubated with 0.1M KOHin methanol for 1 h at 37° C. and separated using the ceramide TLCsystem because the methyl esters migrate near the solvent front underthese conditions.

Results--Rat hepatocytes were selected as a model for the effects offumonisins because F. moniliforme culture materials, now known tocontain the cancer promoting fumonisin B₁ (9,15), are hepatotoxic andhepatocarcinogenic in the rat (4,10,31). Furthermore, sphingosinebiosynthesis de novo by rat hepatocytes is relatively easy to followusing [¹⁴ C]serine, and proceeds via the reactions shown in FIG. 2 (24)like in other cells (23,32).

Fumonisin B₁ (1 μM) caused an almost complete inhibition of [¹⁴C]sphingosine formation by hepatocytes (FIGS. 2 and 3). Similarinhibition occurred when [¹⁴ C]serine and fumonisin B₁ were addedtogether for short term (2 h) or overnight (16 h) incubations and whenthe cells were incubated overnight with fumonisin before adding [¹⁴C]serine (FIG. 3A, the 16 h+2h group). Hence, fumonisin B₁ appears toact fairly quickly, and the inhibition is persistent. The IC₅₀ forinhibition of [¹⁴ C]serine incorporation into [¹⁴ C]sphingosine wasapproximately 0.1 μM for fumonisin B₁ (FIG. 3B). A similar degree ofinhibition was obtained with fumonisin B₂ (FIG. 3A), another mycotoxinoften produced in substantial amounts by F. moniliforme (10).

In contrast, there was no reduction in the radiolabelling ofphosphatidylserine, phosphatidylethanolamine, or phosphatidylcholinefrom [¹⁴ C]serine (Table 1), nor in the mass of these phospholipidsunder conditions where sphingolipid labelling (FIGS. 2 and 3) and mass(Table 1) were reduced significantly. Fatty acid biosynthesis from [¹⁴C]acetic acid was not altered (Table 1).

    ______________________________________                                        Effects of Fumonisin B.sub.1 on selected lipids of rat hepatocytes.sup.a                 μM Fumonisin B.sub.1                                            Parameter.sup.a                                                                            0         1          10                                          ______________________________________                                        A. Radiolabelling                                                                          (Dpm/mg protein × 10.sup.-3) Mean ± SD                  Phosphatidylethanol-                                                                        103 ± 8.3                                                                            163 ± 4.4*                                                                            153 ± 10*                               amine                                                                         Phosphatidylserine                                                                         44.9 ± 9.5                                                                           48.8 ± 2.9                                                                            52.3 ± 3.6                               Phosphatidylcholine                                                                        2270 ± 226                                                                           2640 ± 264                                                                             2740 ± 196*                             Base labile fatty acids.sup.b                                                              2055 ± 261                                                                           2075 ± 54                                                                             2075 ± 103                               B. Mass measurements.sup.c                                                                  (nmol/mg protein) Mean ± SD                                  Phosphatidylethanol-                                                                       44.1 ± 2.9                                                                           44.9 ± 1.4                                                                            44.4 ± 0.9                               amine                                                                         Phosphatidylserine                                                                         13.8 ± 2.8                                                                           15.5 ± 2.6                                                                            12.3 ± 1.9                               Phosphatidylcholine                                                                         118 ± 3.6                                                                            105 ± 1.8                                                                             112 ± 3.4                               Total sphingolipids                                                                         8.9 ± 0.9                                                                            6.7 ± 0.3*                                                                            6.5 ± 0.7*                              ______________________________________                                         .sup.a Hepatocytes were incubated for 16 h with [.sup.14 C]serine and the     concentrations of fumonisin B.sub.1 shown using the conditions described      in FIG. 2. The lipids were extracted, separated by thinlayer                  chromatography, and quantitated as described in the text.                     *The groups that are significantly different (P < 0.05) from the control      (no fumonisin) group are designated by asterisks.                             .sup.b Determined after incubation with 10 μCi of [.sup.14 C]acetic        acid.                                                                         .sup.c Determined by phosphate analyses (27) with the exception of the        total sphingolipids, which were estimated by acid hydrolysis of the lipid     extracts and analysis of the amount of sphingosine by HPLC (28).         

Hence, the inhibition of sphingolipid biosynthesis does not appear to bedue to the inability of the cells to take up [¹⁴ C]serine and toincorporate it into lipids in general, nor to affect the ability of thecell to form the other biosynthetic precursors (i.e., fatty acids).

Although de novo sphingolipid biosynthesis was completely blocked by 10μM fumonisin B₁, there was only a slight reduction in the mass of totalsphingolipids after 1 d (Table 1). This is probably due to the generallyslow turnover of sphingolipids (32). A greater effect was seen when thecells were plated on Matrigel to allow incubation with 1 μM fumonisin B₁for 4 d, which reduced the level of total sphingolipids by about half(i.e., from 4.6±0.7 nmol/dish to 2.1±0.1 nmol/dish). There was nodecrease in the number of viable cells despite treatment with 1 μMfumonisin for 4 d.

There are several potential sites at which fumonisins might affectsphingosine metabolism (FIG. 2); hence, these were investigated further.Fumonisins do not appear to act at the first step of this pathwaybecause even a fairly high concentration (25 μM) fumonisin B₁ did notreduce the activity of serine palmitoyltransferase in vitro (i.e., theactivities in the presence and absence of 25 μM fumonisin B₁ were 88±3and 61±15 pmol 3-ketosphinganine formed/min/mg of microsomal protein,respectively). Furthermore, treatment of hepatocytes with fumonisin B₁followed by assays of this enzyme in disrupted cells revealed noinhibition (i.e., the activities were 7.7±0.2 and 8.2±0.8 pmol/min permg of cellular protein in the presence and absence of 2.5 μM fumonisinB₁, respectively). Inhibition at the second step of the pathway is alsounlikely because 3-ketosphinganine was not seen to accumulate (FIG. 2),and there was no reduction in the formation of [¹⁴ C]sphinganine.

Fumonisins do inhibit at the step where [¹⁴ C]sphinganine is convertedto N-acyl[¹⁴ C]sphinganine because the amount of radiolabel in [¹⁴C]sphinganine increased when hepatocytes were treated with fumonisin B₁(FIG. 4A). This was accompanied by an increase in the mass of freesphinganine (FIGS. 4B and C); when hepatocytes were incubated with 1 μMfumonisin B₁ for 4 d, sphinganine increased 110 fold (i.e., to 1499±18pmol/dish compared to 13.6±0.4 pmol/dish for the control). There wasonly a small reduction in the amount of free sphingosine within thefirst 2 h (FIG. 4B); however, free sphingosine decreased significantlyafter 4 d (i.e., to 52±1 pmol/dish compared to 233±14 pmol/dish for thecontrol). These findings are consistent with the view that the freesphingosine found in hepatocytes is not an intermediate of the de novobiosynthetic pathway (23,24) (hence, will be affected little by shortterm treatment with fumonisin B₁) but arises from the turnover ofcomplex sphingolipids (27,32) and would, therefore, be most affectedwhen there is a decrease in total sphingolipids due to longer termexposure to these compounds.

Inhibition at this step of the pathway was demonstrated directly by invitro assays of ceramide synthase, which has been reported to acylateboth sphinganine and sphingosine (30) (FIG. 5) and by following theconversion of [³ H]sphingosine to [³ H]ceramide by intact cells (FIG.4D). The apparent IC₅₀ for inhibition of this activity in vitro wasapproximately 0.1 μM for fumonisin B₁, but the inhibition may bebiphasic (FIG. 5). All together, these findings demonstrate that theinhibition of ceramide synthase accounts for the disruption of de novosphingolipid biosynthesis by fumonisins, although these compounds mayhave additional effects on other enzymes of sphingolipid metabolism.

The structural basis for this inhibition is due to similarities betweenthe fumonisins and long-chain (sphingoid) bases (FIG. 1), which likelyallow them to be recognized as substrate (or transition state orproduct) analogs by ceramide synthase. The absence of a hydroxyl groupat carbon 1 may alter their orientation in the active site of thisenzyme and preclude acylation or, if acylated, result in an inhibitoryceramide that cannot be removed by addition of a sphingolipid head groupat that position. As shown in FIG. 1, the lack of a hydroxymethyl groupat position 1 is shared by a number of other fumonisin-like compounds,such as the host-specific phytotoxins produced by another fungus,Alternaria alternata f. sp. lycopersici (33); hence, more naturallyoccurring inhibitors of this pathway exist. Data on the Alternariatoxins (AAL toxins) shown in FIG. 1 demonstrates inhibition similar tothat of fumonisins (FIG. 6). Specifically, when Alternaria toxins wereadded to hepatocytes, there was a dose-dependent reduction in theformation of sphingolipids. In other experiments (not shown) we havealso found that Alternaria toxins (AAL toxins) inhibit ceramide synthasein vitro. These compounds are also unable to follow the usual pathway oflong-chain base catabolism, which proceeds via phosphorylation atposition 1 (32), and this may contribute to the persistence of theinhibition.

These findings with rat hepatocytes provide the first identification ofa biochemical target for the action of fumonisins, and demonstrate thatinhibition of de novo sphingolipid biosynthesis in vitro may underliethe hepatotoxicity and hepatocarcinogenicity of this mycotoxin in vivo.The hepatic levels of fumonisins under these conditions are not known;however, if all the fumonisin given in feeding studies with rats (1 to10 mg) (3-5,31) was distributed uniformly in the body, theconcentrations could be between 10 and 100 μM. This is surely an over-(or under-) estimation of the amount that reaches liver in vivo, butcertainly considerably higher than the IC₅₀ for inhibition ofsphingolipid biosynthesis by hepatocytes (0.1 μM). In other studies, wehave noted a 10 and 35% reduction in [¹⁴ C]sphingosine biosynthesis byhepatocytes isolated from two rats given fumonisin B₁ (5 mg/200 g bodyweight) by gavage for just 2 days. There was also a pronounced reductionin the ratio of free [¹⁴ C]sphingosine to [ ¹⁴ C]sphinganine from2.6±0.14 (a typical ratio for control hepatocytes) to 0.5±0.1.

Disruption of this pathway is an attractive mechanism for thepathological effects of fumonisins since sphingolipids are involved inregulating various aspects of cell growth, differentiation, andtransformation (18-20). For example, the degeneration of neuronal cellsseen in equine leucoencephalomalacia can be due to inhibition ofsphingolipid biosynthesis because brain contains high levels ofsphingolipids (17). Also, an accumulation of sphinganine in cellsexposed to fumonisins can lead to cell death since long-chain bases canbe highly cytotoxic (34,35), or to cell proliferation since thesecompounds are mitogenic to some cell types (36) and affect diverse cellsystems (20), including protein kinase C (37), the epidermal growthfactor receptor (38), the Na⁺ /K⁺ ATPase (39) and phosphatidic acidphosphohydrolase (40), inter alia. Thus, although toxic, fumonisins (orrelated compounds) can be therapeutically useful in diseases wheredefects in sphingolipid turnover cause cells to accumulate high levelsof sphingolipids.

Alteration of Serum Sphingolipids upon Dietary Exposure of Ponies toFumonisins, Mycotoxins Produced by Fusarium moniliforme

Materials--Corn screenings were ground in a Romer Mill (Romer Labs,Chesterfield, Mo.) and mixed thoroughly. Samples were taken from eachlot and analyzed for fumonisins by high-performance liquidchromatography (HPLC) and thin-layer chromatography as is known in theart as previously described in (15). If the amounts of fumonisin B1showed greater than 10% variation (relative standard deviation in 8samples from each 25 kg lot), the lot was mixed, sampled, and analyzed asecond time or until variance was less than 10%. Using the meanfumonisin B level, individual rations were prepared daily by adding thecorn screenings to pellets (NADC Horse Ration, Code 560, Purina Mills,St. Louis, Mo.) and molasses (Promolas Easy Mixer Supreme, PacificMolasses Co., San Francisco, Calif.) to yield the fumonisinconcentrations cited in the text. In general, the ration contained 60%pellets, 10% molasses, and 30% corn screenings. The control dietconsisted of pellets plus molasses.

All portions of the diet were subjected to general screens for toxicantsby gas chromatography/mass spectroscopy and inductively coupled plasmaspectroscopy, and assayed for chlorinated hydrocarbon pesticides,organophosphate pesticides, carbamate pesticides, aflatoxins,zearalenol, T-2 toxin, diacetoxyscirpenol, deoxynivalenol, 15-acetyldeoxynivalenol, fusarenone-x, zearalenenone, HT-2 toxin, T-2 tetrol,sterigmatocystin, cyclopiazonic acid, ochratoxin A, gliotoxin,sporodesmin, and griseofulvin. In addition, the hay, molasses, andpellets were tested for fumonisin B1 and B2.

Feeding protocols--Clinically normal ponies of varying age and sex wereconditioned on the control diet for 21 days prior to the trial. Horse341 was a 6 year old gelding (173-193 Kg), 299 was a 12 year old mare(205-215 Kg), 164 was a 12 year old mare (206-290 Kg) and 166 was a 16year old mare (143-164 Kg). During the conditioning period andthroughout the trial, all ponies received alfalfa hay by free choice.The test groups were fed varying levels of fumonisin (15 to 44 ppm) forvarying lengths of time. The individual unconsumed rations were measureddaily; at certain times, the ponies consumed variable portions of therations, and these have been indicated in the figures.

Analysis of serum long-chain bases--Blood was drawn approximately twicea week, centrifuged to obtain serum, and aliquots were frozen foranalyses of free sphingosine and sphinganine by HPLC as is known in theart as described in (28). To quantitate total sphingolipids in somesamples, the extracts were acid hydrolyzed before preparing the samplesfor HPLC.

Clinical chemistry, evaluation, and tissue collection--The followingclinical chemistry parameters were also analyzed with blood: totalbilirubin, direct bilirubin, urea nitrogen, alkaline phosphatase,AST/SGOT, creatine kinase, gamma glutamyl transaminase, and bile acids.The animals were observed intermittently throughout each day andbehavior changes were noted. Animals that were euthanized or diedunderwent a routine necropsy, and brain, liver, and kidney were preparedfor microscopic study.

Results--Analyses of sera from three ponies that had not been exposed tofumonisin contaminated feed produced HPLC profiles like the one shown inTable 2.

                  TABLE 2                                                         ______________________________________                                        PEAK NO.  TIME        AREA      CONC                                          ______________________________________                                        1         1.088        20366    0.0898                                        2         1.733       22118978  97.5714                                       3         2.413       134152    0.5918                                        4         2.944        94677    0.4176                                        5         3.971        4455     0.0197                                        6         4.2          2677     0.0118                                        7         4.47          773     0.0034                                        8         5.21         2416     0.0107                                        9         5.483        2141     0.0094                                        10        6.283        11185    0.0493                                        11        8.28         3517     0.0155                                        12        13.688      274192    1.2095                                        TOTAL             22669520  100                                               ______________________________________                                    

Sphingosine elutes at 6.3 min and, for this sample, sphinganine (at 8.3min) can also be seen clearly. In some instances, the amount ofsphinganine was too low to detect above background. The compound elutingat 13.7 min is the internal standard (C20-sphinganine) that is added toaid extraction and quantitation of the long-chain bases (28).

Data for sera collected from three ponies at intervals of 2 weeks to 9months are given in Table 3.

                  TABLE 3                                                         ______________________________________                                        Levels of free long-chain bases in sera from four ponies                      given feed thought to be free of contamination by fumonisins.                 Days after  Sphingosine Sphinganine                                                                              Ratio                                      initial sampling                                                                          pmol/ml     pmol/ml    Sa/So                                      ______________________________________                                                  Pony #299                                                           0           46.8 ± 1.2                                                                             13.6 ± 0.4                                                                            0.29                                       14          39.3 ± 1.6                                                                             10.9 ± 1.2                                                                            0.28                                       65          107 ± 18 22.9 ± 6.0                                                                            0.19                                       248         59.3 ± 6.0                                                                             16.4 ± 6.4                                                                            0.26                                                 Pony #164                                                           0           118 ± 5  22.6 ± 4.6                                                                            0.19                                       2            97 ± 10 32.4 ± 8.0                                                                            0.32                                       7           63 ± 1   27.3 ± 1.4                                                                            0.43                                       22          42 ± 2   11.7 ± 4.0                                                                            0.26                                                 Pony #166                                                           0            82 ± 24 22.2 ± 1.8                                                                            0.25                                       2           33 ± 1   20.7 ± 0.2                                                                            0.63                                       7           54 ± 1   20.8 ± 2.0                                                                            0.40                                       22          43 ± 4   13.7 ± 0.6                                                                            0.32                                       ______________________________________                                    

Sphingosine is the predominant free long-chain base in serum from allponies. The mean ±SD for all of the analyses (n=12) were: 65±29 pmolsphingosine/ml serum and 19.5±6.5 pmol sphinganine/ml serum. There weresome variability in the amounts of these compounds over time, althoughthis was seen less in sphinganine than sphingosine; furthermore, thesphinganine to sphingosine ratio (0.32±0.12) was fairly constant. Thislatter parameter may be useful because these compounds are sometimesdifficult to extract efficiently; hence, a ratio of two structurallyrelated species (i.e., sphinganine and sphingosine versus theC20-sphinganine standard or sphinganine versus sphingosine) will be moreaccurate than absolute measurements of the mass of a single compound.

Table 4 illustrates a serum sample from the same pony as in Table 2after exposure to fumonisin-contaminated feed.

                  TABLE 4                                                         ______________________________________                                        PEAK NO.  TIME       AREA       CONC                                          ______________________________________                                        1         1.091      23970      0.1095                                        2         1.733      21355228   97.5859                                       3         2.412      74417      0.3401                                        4         2.945      97222      0.4443                                        5         4.166      17641      0.0806                                        6         5.09       11076      0.0506                                        7         5.507       2952      0.0135                                        8         5.883       2220      0.0101                                        9         6.328      47332      0.2163                                        10        7.627       1052      0.0048                                        11        8.326      70389      0.3217                                        12        13.761     180023     0.8226                                        TOTAL            21883512   100                                               ______________________________________                                    

Difference in the HPLC profile are evident both on the magnitude of thepeaks and the relative amounts of sphingosine and sphinganine. Thelevels of sphingosine and sphinganine are summarized in Table 5 for twoponies given 44 ppm fumonisin B1, a level that is often toxic andresulted in ELEM in both of these animals (pony 164 died after 10 daysand pony 166 was euthanized on day 45).

                  TABLE 5                                                         ______________________________________                                        Levels of free long-chain bases in sera from two ponies                       given feed contaminated with fumonisin B1 at 44 ppm                           Day of Sphingosine                                                                              Sphinganine                                                                              Ratio  AST/SGOT                                  Trial  pmol/ml    pmol/ml    Sa/So  Units                                     ______________________________________                                        Pony #164.sup.1                                                               -1     42 ± 2  11.7 ± 4.0                                                                            0.26   248                                       2      117 ± 7 70.2 ± 4.2                                                                            0.60   235                                       7      152 ± 13                                                                              253 ± 22                                                                              1.67   357                                       10     226 ± 20                                                                              283 ± 18                                                                              1.25   4000                                      Pony #166.sup.2                                                               -1     43 ± 4  13.7 ± 0.6                                                                            0.32   210                                       2      43 ± 6  37.2 ± 0.2                                                                            0.87   199                                       7       66 ± 16                                                                              62 ± 8  0.95   215                                       10     92 ± 4  179 ± 8 1.92   1470                                      13     100 ± 8 174 ± 18                                                                              1.72   2080                                      ______________________________________                                         .sup.1 Pony was described as nervous and off feed from day 5; by day 9 wa     blind and down, and on day 10 had full clinical signs of ELEM and died        that evening.                                                                 .sup.2 Pony was described as lethargic from day 5; off feed from day 8;       later put back on full feed and exhibited CNS signs; was euthanized on da     45.                                                                      

By day 2, pony 164 exhibited elevations in sphingosine and sphinganine;however, the increase in sphinganine was greatest (6-fold compared tothe level immediately prior to feeding fumonisin) and this was reflectedin an increase in the ratio of sphinganine to sphingosine, whichdoubled. By day 7, sphinganine had increased 22-fold and sphingosine was3.6-fold compared to the initial level. The pronounced increase insphinganine over sphingosine gave a sphinganine to sphingosine ratio(1.67) that is unprecedented in any previous analyses of long-chainbases in mammalian systems--except our above studies of the effects offumonisins on hepatocytes.

Pony 166 also showed an increase in sphinganine (2.7 fold), but notsphingosine, after only two days on contaminated feed, the earliesttimepoint examined. By day 7, sphingosine and sphinganine were bothelevated, but sphinganine had increased to the level of sphingosine. Byday 10, pony 166 showed elevations in sphingosine (2-fold), sphinganine(13-fold), and AST/SGOT (7-fold) that indicate significant hepaticinjury.

Another pony (#212) was initially given 44 ppm of fumonisin, but lesswas eaten because of variable consumption of the feed and because thefumonisin was withheld for two weeks. The results for this animal areshown in FIG. 7. As for the other two ponies, there was a rapid increasein free long-chain bases that slightly preceded the clinical signs oftoxicity (i.e., elevated AST/SGOT). Within 2 days the sphingosine levelhad nearly doubled over the amount the day prior to administration ofcontaminated feed (i.e., from 49.1±5 pmol to 147±5 pmol); sphinganinewas also elevated but the fold increase could not be calculated becausesphinganine was not detectable in serum from this pony before the trial.By 7 days, sphingosine had increased another 2-fold, but sphinganine was6.7 fold higher than the level at day 2, and now exceeded the level ofsphingosine by 1.9 fold. After day 10 of the study, both sphinganine andsphingosine decreased, although the ratio remained elevated above thatbefore exposure to fumonisin. The decrease was during the period wherethe pony consumed variable amounts of the contaminated feed (i.e., fromday 5 through 31) and when the feed was withdrawn all together. Thecontaminated feed was provided again on day 45 and was consumed variablyuntil termination of the study on day 97. Restoration of the fumonisinresulted in small increases in sphingosine and sphinganine, but mostprominently in the sphinganine to sphingosine ratio of 2 to 3. Thisbehavior suggests that serum sphingosine and sphinganine, and mostespecially their ratio, increases after fumonisin exposure, returns tomore normal levels upon removal of (or more variable consumption of)contaminated feed, and increases again when the animals are reexposed tofumonisin in the diet.

The fourth pony (#341) was given lower amounts of fumonisin-contaminatedfeed as shown in FIG. 8. During the first 130 days at 15 ppm fumonisinB1, the levels of free sphingosine and sphinganine fluctuated with nonoticeable pattern (data not shown); however, when the data areexpressed as the ratio of these compounds, there was a fairly consistentelevation over the initial value. Although the initial ratio is based ona single serum sample at the start of the experiment, it was similar tothat of the other animals studied.

When pony 341 was taken off the fumonisin between day 130 and 160, theratio dropped to a level that was below the starting level. Fumonisinfeeding was resumed at 22 ppm on day 161, and there was a rapid increasein the sphinganine to sphingosine ratio, and the radio declined againwhen the pony was taken off fumonisin between days 175 and 182.Reintroduction of 22 ppm fumonisin B1 on day 182 was accomplished by arapid increase in the ratio to >1.5, which culminated in a precipitousfall on day 235. Later that day the pony died with the symptoms ofequine leucoencephalomalacia, which was later confirmed by biopsy. Thisfinal decrease in the ratio was due to a greater than two-fold increasein sphingosine (i.e., from 163 to 418 pmol/ml between day 231 and 235,rather than to a reduction in sphinganine (which also increased from 256to 532 pmol/ml.

The upper panel of FIG. 8 shows serum AST/SGOT of pony 341 over thisperiod. The activities were essentially unaffected until between day 224and 231. Other markers of liver function were also unaltered until thisperiod. Thus, changes in the free long-chain bases appeared much earlier(at least a month or more) before exposure to fumonisins was detectableby elevations in liver enzymes.

If the elevation in sphinganine is due to inhibition of de novosphingolipid biosynthesis, this might also be reflected in a reductionin the amounts of examined with sera from several of the ponies as shownin Table 6.

                  TABLE 6                                                         ______________________________________                                        Levels of long-chain bases liberated from the total serum lipids              upon acid hydrolysis to cleave more complex sphingolipids                            Sphingosine    Sphinganine   AST/                                      Day of pmol/ml        pmol/ml       SGOT                                      Trial  Total (-free)  Total (-free) units                                     ______________________________________                                        Pony 164                                                                      -20    3400 ± 266 (ND)                                                                           203 ± 18 (ND)                                                                            270                                       -15    2670 ± 678 (2630)                                                                         139 ± 22 (127)                                                                           224                                         2    621 ± 99 (504)                                                                            169 ± 30 (99)                                                                            235                                         7    254 ± 32 (102)                                                                            156 ± 48 (0).sup.2                                                                       357                                        10    2960 ± 585 (2740)                                                                          786 ± 234 (503)                                                                         4000                                      Pony 166                                                                      -22    3620 ± 452 (3580)                                                                         123 ± 23 (103)                                                                           215                                       -15    4430 ± 409 (4380)                                                                         93 ± 9 (70)                                                                              180                                        -1    2340 ± 694 (2290)                                                                         211 ± 69 (197)                                                                           210                                         2    1020 ± 313 (974)                                                                          131 ± 40 (94)                                                                            199                                         7    950 ± 154 (985)                                                                           206 ± 18 (144)                                                                           215                                        10    634 ± 192 (540)                                                                           212 ± 28 (34)                                                                            1470                                       13    1740 ± 150 (1640)                                                                         823 ± 54 (650)                                                                           2080                                      Pony 208                                                                      -22    6940 ± 88 (6610)                                                                          267 ± 11 (235)                                                                           419                                       -15    4950 ± 163 (4750)                                                                         178 ± 3 (153)                                                                            327                                        -1    5540 ± 42 (5270)                                                                          218 ± 18 (279)                                                                           305                                         2    3490 ± 358 (3190)                                                                         387 ± 26 (107)                                                                           344                                         6    2390 ± 388 (2030)                                                                         718 ± 75 (60)                                                                            2130                                       10    4450 ± 10 (3460)                                                                          2040 ± 70 (200)                                                                          1240                                       13     15970 ± 1576 (15040)                                                                      9990 ± 379 (9604)                                                                       1010                                      ______________________________________                                         .sup.1 The description of the ponies is given in the text and Table 3.        Data is not given for a few of the days of feeding because there was too      little sample available to conduct the analysis.                              .sup.2 These values underestimate the contribution of free longchain base     because they are somewhat unstable under the acid hydrolysis conditions. 

For these analyses, the lipids were extracted and then hydrolyzed toliberate the free long-chain bases which were analyzed by HPLC. Theresults (Table 6) show that the levels of total (complex) sphingolipidswere reduced by 50 to 95% during the early times after feedingfumonisin, but that the levels increased again when there was also anelevation in hepatic enzymes. As discussed earlier, this increase mayreflect the release of membrane sphingolipids into circulation upon cellinjury.

Since pony 164 was clearly undergoing rapid tissue injury (as reflectedin the marked elevation in sphingosine was due to the turnover ofsphingolipids in injured tissues. This is, again, consistent with amechanism whereby a block at the level of acylation of newly synthesizedsphinganine occurs as described above. That study showed that inhibitionof ceramide synthase results in accumulation of sphinganine as anintermediate of de novo sphingolipid biosynthesis. The free sphingosine(and some sphinganine) occurs from the turnover of existing complexsphingolipids; hence, this will be affected somewhat as fumonisinsinhibit the reacylation of this long-chain base. In both the magnitudeand the speed with which changes appeared, the increase in serumlong-chain bases was an earlier indicator of exposure to fumonisins thanelevations in AST/SGOT for both of these ponies. Hence, changes in thesphinganine to sphingosine ratio can detect exposure of ponies tofumonisin by 1 to 2 months earlier than liver function tests.

Synthesis of Fumonisin and Fumonisin Analogs

The following more particularly describes the synthesis of fumonisin andfumonisin analogs (see FIG. 1), which are intended as illustrative onlysince numerous modifications and variations therein will be apparent tothose skilled in the art. Numeral references following a particularcompound refer to the structures found in FIG. 9.

The synthesis of the fumonisin analog 2-aminooctadeca-3,5-diol fromL-alanine methyl ester hydrochloride (1) is given below. Obviously, aparticular fumonisin or other fumonisin analogs can be formed utilizingthis general synthesis by the addition of the appropriate Grignardreagent to compound 4 and, if necessary, functionalization of oxygen ornitrogen atoms contained in that Grignard reagent at the appropriatetime to attach mono- or polycarboxylate moieties to the tail of thecompound. Ester or ceramide couplings of this type can be readilyaccomplished using a variety of reagents well-known to those skilled inthe art. Furthermore, the X and Y substituents, as shown in thefumonisin analog of FIG. 1, can be added by methods known in the art.introduction of the V-substituent can be readily accomplished using ahydride opening of an epoxide (e.g., 6 of FIG. 9) to obtain V=H) or anepoxide opening with a variety of oxygen nucleophiles to ultimatelyobtain V=OH. By controlling both the stereochemistry of the epoxidation(e.g., 5a→6 of FIG. 9) and the regiochemistry of the subsequent epoxideopening with hydride or oxygen nucleophiles, the preparation of allpossible variations of V and Y=H or OH can be achieved. Because the headgroup is likely to be an important factor in modulating the biologicalactivity of the compound in question, modifications of this type mayprove to be important.

L-Alanine methyl ester hydrochloride (1) (1.0 eq, 179.1 mmol, 25 gm) wasdissolved in 500 ml of dry N, N-dimethylformamide (DMF) under a nitrogenatmosphere. Distilled triethylamine (2.5 eq, 447.2 mmol, 45.3 gm) anddi-t-butyl dicarbonate ((BOC)₂ O) (1.1 eq, 197.4 mmol, 43 gm) were thenadded and the reaction mixture was heated and stirred at 60° C. for 1hour and at room temperature for an additional 4 hours. The DMF was thenevaporated in vacuo and the remaining residue was dissolved in ethylacetate, cooled to 5° C., washed with 10% citric acid, saturated NaHCO₃,and brine, dried over anhydrous MgSO₄, filtered, and the solvent wasevaporated in vacuo. The N-BOC-protected ester 2 was purified via flashcolumn chromatography and a colorless oil was obtained in 88% yield.

Ester 2 (1.0 eq, 49.3 mmol, 10 gm) was dissolved in dry toluene (400 ml)and cooled to -78° C. under a nitrogen atmosphere. Diisobutylaluminumhydride (DIBAL-H) (1.4 eq, 69.0 mmol) was then added slowly and thereaction monitored by TLC. After 3.5 hours, the reaction was quenched at-78° C. with ethanol and allowed to warm to room temperature. The turbidsolution was filtered through silica gel and the aluminum salts werethoroughly washed with ether. Rotary evaporation of the solvent yieldedthe product (3) (82% yield) as a white solid and the aldehyde waspurified by flash column chromatography.

Aldehyde 3 (1.0 eq, 17.9 mmol, 3.1 gm) and triphenylphosphoranylidineacetaldehyde (2.2 eq, 39.4 mmol, 12.1 gm) were dissolved in 250 ml ofdry benzene and refluxed under a nitrogen atmosphere for 2 hours.Benzene was evaporated in vacuo and the crude product (brown solid) waspurified via flash column chromatography. The unsaturated aldehyde 4 wasisolated in 72% yield and used immediately.

An excess of magnesium turnings was placed in a 3-neck round bottomflask and flame-dried under a vacuum. After cooling, 125 ml of ether wasadded followed by 1-bromotridecane (2.2 eq, 28.8 mmol, 7.6 gm) and themixture was stirred under reflux for 24 hours. Aldehyde 4 (1.0 eq, 13.1mmol, 2.6 gm) was dissolved in 100 ml of ether under a nitrogenatmosphere and cooled to 0° C. The Grignard reagent was then cannulatedinto the aldehyde solution and allowed to warm up to room temperaturefor 2 hours. The reaction was then quenched with saturated NH₄ Clsolution and the organic layer was washed with brine, dried over MgSO₄,and concentrated in vacuo. The diasteriomeric mixture of alcohols (5)were separated via flash column chromatography and isolated in 86%yield.

Allylic alcohol 5a (1.0 eq, 0.63 mmol, 240 mg) was dissolved in 10 mlCHCl₂ and 10 ml of 0.5M NaHCO₃. meta-Chloroperbenzoic acid (1.1 eq, 0.69mmol, 120 mg) was added slowly and the reaction was stirred overnight.The organic layer was then washed with brine, dried over MgSO₄ andconcentrated in vacuo. The mixture of diasteriomeric epoxides (6) wereseparated by flash column chromatography in 90% yield.

Epoxide 6a (1.0 eq, 72.5 mmol, 30 mg) was dissolved in 5 ml of dry THFand cooled to 0° C. under a nitrogen atmosphere.Bis(2-methoxyethoxy)aluminum hydride (2.5 eq, 0.19 mmol) was then addedand the reaction mixture was allowed to warm up overnight. The reactionwas quenched with ethanol, filtered over celite and concentrated invacuo to produce diol 7 in 76% yield after purification. Sodiumtriacetoxyborohydride can also be utilized to produce diol 7 at thisstep.

Carbamate 7 (1.0 eq, 124 mmol, 50 mg) was dissolved in 25 ml of ethylacetate and cooled to 0° C. under a nitrogen atmosphere. Dry HCL gas (>3eq) was bubbled through the solution and allowed to stir for 6 hours at0° C. as determined by TLC. The reaction was then basified to pH 9 with10% NH₄ OH and the organic layer washed with brine, dried over anhydrousMgSO₄, and concentrated in vacuo. Flash column chromatography producedthe amino diol 8 as a white solid in 78% yield.

With reference to the definition of fumonisin analogs given above, theSpacer Groups can be added to the various head groups in two differentfashions. The first involves a Wittig (or Wadsworth/Emmons/Horner)olefination in which an ylide containing a terminal oxygen, nitrogen orcarboxyl functional group is allowed to react with an aldehyde. Examplesof the types of ylides used are given by the structure: ##STR1## wheren=1-20 (straight or branched chain and X=O-Protecting Group,NH-Protecting Group, NR-Protecting Group, CO₂ H (Li).

The second approach involves an acetylide addition to an aldehyde. Thetypes of acetylides which are used are shown below:

    M.tbd.(CH.sub.2).sub.n --X

where n=1-20 (straight or branched chain; X=O-Protecting Group,NH-Protecting Group, NR-Protecting Group (wherein R=a straight orbranched chain C₁ -C₆ alkyl), CO₂ H(M); and M=Li, Na, K, etc. As usedherein, "a Protecting group" refers to a moiety that provides protectionfor the corresponding alcohol or amide until deprotection is desired.This protection/deprotection process is well-known in the art asdiscussed in (41).

FIG. 10 shows various reaction schemes that can be employed to controlthe stereochemistry of the 2-aminopenta-3,5-diol head group during thesynthesis of a fumonisin or fumonisin analog. FIG. 11 shows variousreaction schemes that can be employed to control the stereochemistry ofthe aminotriol head groups present in Alternaria toxins or fumonisinanalogs similar to Alternaria toxins.

Although the present processes have been described with reference tospecific details of certain embodiments thereof, it is not intended thatsuch details should be regarded as limitations upon the scope of theinvention except as and to the extent that they are included in theaccompanying claims.

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What is claimed is:
 1. A method of detecting the consumption of afumonisin, or an analog thereof, in a subject comprising detecting, in asample from the subject, the presence of inhibition of ceramidesynthease, the presence of the inhibition of ceramide synthaseindicating the consumption of a fumonisin or fumonisin analog.
 2. Themethod of claim 1, wherein the inhibition of ceramide synthase isdetected by detecting an increase in sphinganine in the sample from thesubject.
 3. The method of claim 1, wherein the inhibition of ceramidesynthase is detected by detecting a decrease in ceramide in the samplefrom the subject.
 4. The method of claim 1, wherein the inhibition ofceramide synthase is detected by detecting an increase in the amount ofsphinganine relative to the amount of sphingosine in the sample from thesubject.
 5. The method of claim 1, wherein the fumonisin is fumonisinB₁.
 6. The method of claim 1, wherein the fumonisin is fumonisin B₂. 7.The method of claim 1, wherein the fumonisin analog is an Alternariatoxin.
 8. The method of claim 1, wherein the subject is a human.
 9. Themethod of claim 1, wherein the subject is a horse.